In view of that conjugated polymers (CPs) are an attractive option for constructing high-sensitive Cr2O72− sensors but suffer from lacking a general design strategy, we first proposed a rational structure design of CPs to tailor their sensing properties while validating the structure-to-performance correlation. Short side chains decorated with N and O atoms as recognition groups were instructed into fluorene to obtain monomers Fmoc-Ala-OH and Fmoc-Thr-OH. Additionally, their polymers P(Fmoc-Ala-OH) and P(Fmoc-Thr-OH) were obtained through electrochemical polymerization. P(Fmoc-Ala-OH) and P(Fmoc-Thr-OH) with high polymerization degrees have an excellent selectivity towards Cr2O72− in comparison to other cations and anions. Additionally, their limit of detection could achieve 1.98 fM and 3.72 fM, respectively. Especially, they could realize the trace detection of Cr2O72− in agricultural products (red bean, black bean, and millet). All these results indicate that short side chains decorated with N and O atoms functionalizing polyfluorene enables the ultra-trace detection of Cr2O72−. Additionally, the design strategy will spark new ideas for the construction of highly selective and sensitive Cr2O72− sensors.